Composition For Treatment Of Articular Cartilage Damage

ABSTRACT

Disclosed is a composition for the treatment of cartilage or bone damage or loss or defect. The composition comprises mesenchymal stem cells separated from umbilical cord blood and/or mesenchymal stem cells proliferated and/or differentiated. The composition also comprise chondrocytes and/or chondroblasts, or osteocytes and/or osteoblasts, differentiated from the mesenchymal stem cells separated from the umbilical cord blood.

This is a continuation application of U.S. Ser. No. 10/485,816 (allowed)filed on Apr. 5, 2004, which is a national stage application under 35U.S.C. 371 of PCT/KR02/01552 filed on Aug. 14, 2002, which claimspriority from Korean patent application 2001/49147 filed on Aug. 14,2001, and from Korean patent application 2001/49148 filed on Aug. 14,2001, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to a composition for the treatment ofarticular cartilage damage or loss or defect.

2. Description of the Prior Art

The articular cartilage damage induces pain in the articular region,fault of the articular movement, etc., and lowers the quality of life aswell as productivity. Particularly, it is difficult to treat completelythe articular cartilage damage since the natural healing power is verylow and it is connected to the damage to the entire articulation as itis progressed continuously once it occurs.

The methods of treatment of articular cartilage damage developed up tothe present time include chondroplasty, osteochondral transplantation,autologous chondrocyte transplantation, etc.

Chondroplasty is the most generally used method among theabove-described methods. The arthroscopic operation which is therepresentative method is a method in which diagnosis and operation maybe performed simultaneously while magnifying and observing inside of thearticulation through a TV monitor by inserting an arthroscope, on whicha small-sized camera is mounted, into the articular cavity through asmall hole of less than 1 cm.

The above method is advantageous in that it is possible to reduce painand burden of a patient since it is not necessary to have a directincision of articulation and it is possible to cure immediately afterminor damages to tissues are observed through the arthroscope. However,this chondroplasty is not satisfactorily effective in view of itsfunctional aspects since the fibro-cartilage, not hyaline cartilage thatis necessary for the articulation actually, is produced mainly.

In the meantime, osteochondral transplantation is a method of producinghyaline cartilage by collecting both of the cartilage and sub-cartilageportions produced already in the normal section of a patient andtransplanting them in the damaged cartilage section by making holesproperly. This method has been successful in some patients. However,this method may not be said to be a perfect treatment method because ofa problem of having cracks between the transplanted portion and originaltissues, and is not a general method in that it may be applied to onlythe patients who are able to be subject to autologous transplantation.And the above method may not be operated if the damaged portion is largesince the donated portion is limited, and it is likely thatcomplications occur in the donated portion. Also, the process ofoperation is comparatively complicated, and sometimes it is not possibleto perform arthroscopic operation. In other words, the above method hasweaknesses such as a new pain in the donated portion and may incurcomplications such as a slow rehabilitation including pain, fracture,bleeding, and scar after operation.

Autologous chondrocyte transplantation that has been started to beemployed recently is a method of filling the cartilage portion damagedby proliferation of these cells by obtaining chondrocytes from cartilagetissues collected from the normal portion of a patient, culturing andgrowing them as much as they are needed externally, securing a space byusing periosteum and injecting them to the damaged portion of cartilagealong with the culture medium.

Compared to the osteochondral transplantation method in which alreadyproduced cartilage tissues are injected to the damaged portion, there isa more possibility of reproducing hyaline cartilage as the transplantedportion is fused comparatively well with the normal portion since thedamaged portion is filled with transplanted chondrocytes as they areproliferated directly in the damaged portion. However, there are stillpain, aftereffects, and economical burden of a patient eventually due tothe operation of twice and the process of operation is also complicatedand difficult since it is necessary to perform operation when collectingchondrocytes and when transplanting those cultured externally aretransplanted to the portion of articular cartilage damage.

And there are problems with chondrocytes in that it takes a considerableamount of time until as much as the cells that are necessary fortransplantation during external culturing of cells are obtained as theproliferation and growth of the cells collected are not active since thechondrocytes collected are obtained from fully grown adults in mostcases; the treatment itself could not be accomplished if the cells losethe ability to proliferate at all; and the form of expression of cellsis changed since chondrocytes are cultured externally. And it is ofconcern that the life of cartilage made by culturing again fully growncells would not be long. And in case of the second operation,complications such as pain, scar, etc. after operation are inevitablesince it is necessary to perform a serious incision since noarthroscopic operational methods have been developed yet.

It has been reported that there has been a method of obtainingmesenchymal stem cells (MSCS) that are precursor cells of chondrocytesand osteoblasts from mesenchymal tissues such as autologous bonemarrows, muscles, skin, etc., proliferating them ex vivo, and injectingthem into the portion of articular cartilage damage together withpolymers.

It is shown that the cell proliferation ability in the method oftreatment of cartilage damage by using mesenchymal stem cells obtainedfrom grown individuals as described in the above is somewhat higher thanthat of the autologous chondrocyte transplantation method since moreundifferentiated cells are obtained and cultured ex vivo. However, theabove-described method still shows a weak ability to proliferate cellsfor the treatment of various ways of cartilage damage fully. Also, theabove-described method is required to have a difficult process ofcollection of bone marrows, and is limited in that construction of aninfrastructure such as bone marrow storage banks, etc. is weak.

As described in the above, the methods of treatment of articularcartilage damage developed up to the present time have been problematicin view of their operational processes and effects.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide acomposition for the treatment of articular cartilage damage or loss ordefect composed of cellular components separated, proliferated, and/ordifferentiated from the umbilical cord blood, and biocompatible polymersthat can have superior effects of treatment of articular cartilagedamage through relatively simple operations.

The composition for the treatment of articular cartilage damage of thepresent invention is characterized by that it is composed of cellularcomponents separated, proliferated, and/or differentiated from theumbilical cord blood, and their media. Another composition for thetreatment of articular cartilage damage or loss or defect of the presentinvention is characterized by that it is composed of the above cellularcomponents, their media, and biocompatible polymers. Still anothercomposition for the treatment of articular cartilage damage or loss ordefect of the present invention is characterized by that it is composedof cellular components separated or differentiated from the umbilicalcord blood, and biocompatible polymers.

The cellular components of the composition for the treatment ofarticular cartilage damage or loss or defect of the present inventionare one or more cellular components such as mesenchymal stem cellsand/or mesenchymal stem/progenitor cells separated and/or cultured fromthe umbilical cord blood, precursor cells that are differentiated fromthe above mesenchymal stem cells and/or stem/progenitor cells,chondrocytes and/or osteoblasts that are differentiated from the aboveumbilical cord blood-derived mesenchymal stem/progenitor cells.

In the composition for the treatment of articular cartilage damage ofthe present invention, the umbilical cord blood which is the originatingtissue of cellular components is defined to be the blood collected fromthe umbilical vein connecting placenta and fetus.

Among the cellular components of the composition for the treatment ofarticular cartilage damage of the present invention, mesenchymal stemcells separated from the umbilical cord blood may be differentiated intomesenchymal tissues such as bones, cartilage, fatty tissues, muscles,tendon, etc. under proper conditions for differentiation since they aremultipotent contrary to typical stromal cells of bone marrows. Also,mesenchymal stem cells originated from the umbilical cord blood are thecells that may be proliferated under proper conditions without beingdifferentiated into specific cells or tissues since they have theability to self-renewal.

Among the cellular components of the composition for the treatment ofarticular cartilage damage of the present invention, precursor cellsinclude all those that may be obtained during the process ofdifferentiation of mesenchymal stem cells of the umbilical cord bloodinto chondrocytes or osteocytes, chondroblasts, osteoblasts, etc. amongthe cells originated from mesenchymal stem cells of the umbilical cordblood.

The cellular components of the composition for the treatment ofarticular cartilage damage of the present invention may have a muchsuperior ability to proliferate and differentiate since they are thecells originated from younger cells compared to the cells originatedfrom cells including mesenchymal stem cells separated from adult tissuessuch as bone marrows, muscles, skin, etc.

In the collection and acquisition of originating tissues of the cellularcomponents of the composition for the treatment of articular cartilagedamage of the present invention, it is much easier to collect thecellular components than to collect cells from adult tissues such asbone marrows, etc. that require for operative procedures.

Moreover, it is easy to find a donor for the umbilical cord blood sincebanking or storing of umbilical cord blood is more feasible at birth andthe stored umbilical cord blood can be used easily after thawing.Routine storing of bone marrow like umbilical cord blood is impossible.

And since the cellular components of the composition for the treatmentof articular cartilage damage of the present invention are the cells ofwhich major histocompatibility antigen HLA-DR (Class II), which is themost important cause of rejection reaction in interpolation or organtransplantation, is not expressed, the autologous umbilical cord bloodas well as allogenic umbilical cord blood may be used in that it ispossible to avoid bringing about or minimize immune reactions such asrejection reaction, etc. that may be the problem of the conventionaltransplantation operation.

The media of the composition for the treatment of articular cartilagedamage of the present invention is for suspending cellular components.Generally used cell culture media such as McCoys 5A media (Gibco),Eagle's basal media, CMRL media, Glasgow minimum essential media, Ham'sF-12 media, Iscove's modified Dulbecco's media, Liebovitz' L-15 media,RPMI 1640 media, etc. may be used.

In the present invention, if necessary, one or more secondary componentsmay be added to the cell culture media. That is, one or more componentsselected from the sera of a fetal calf, horse, human, etc.; antibioticssuch as Penicillin G, gentamycin, streptomycin sulfate, etc. forpreventing contamination of microorganisms; antifungal agents such asamphotericin B, nystatin, etc. may be used.

Biocompatible polymers of the composition for the treatment of articularcartilage damage of the present invention are characterized by havingone or more characteristics among the biocompatibility, biodegradationproperty and ability to enhance nutrition of cells and ability toenhance formation of intercellular substrate. Biocompatible polymers ofthe composition for the treatment of articular cartilage damage of thepresent invention have the semi-solid or gel-like property to the degreethat is similar to that of ointments or pastes as well as the mechanicalstrength and flexibility to the degree that are proper for cellulartransplantation and regeneration of cartilaginous tissues or bone.

Accordingly, the composition for the treatment of articular cartilagedamage of the present invention is able to accelerate proliferation anddifferentiation of chondrocytes that are transplanted together as it islocated at the damaged region continuously while maintaining a constantshape once it is transplanted and seeking for convenience in operationsince its shape may be changed readily to conform to various3-dimensional geometry that may be shown in the damaged region ofcartilage.

Biocompatible polymers that may be used for the composition for thetreatment of articular cartilage damage of the present invention may beone or more components selected from natural polymers such as proteins,polysaccharides, etc.; synthetic polymers comprised of hydroxy acids ortheir derivatives; and organic polymers forming the 3-dimensionalscaffold or lattice structure according to chemical binding and theirderivatives and transformed compounds. For example, the proteins amongnatural polymers include fibrin, gelatin, and collagen; saccharidesamong natural polymers include hyaluronic acid, etc.; synthetic polymersinclude polyphosphazine, polyacrylate, polyglactic acid, andpolyglycolic acid; and organic polymers include pluronic acid, alginicacid and its salts, etc.

It is possible to promote the mechanical strength and flexibility of thecomposition for the treatment of articular cartilage damage of thepresent invention by adding the chitosan fiber.

If a polymer is used singly in the present invention, it is possible toadjust its concentration according to the molecular weight andcharacteristics of each polymer component. The final content of pluronicacid after it is mixed with cells when it is used singly is 30%, and itis preferable to make the final content of hyaluronic acid 3-4% after itis mixed with cells when it is used alone.

When using polymers mixedly in the present invention, it is preferableto use those that are made by mixing them in such a way that the finalcontent of pluronic acid after it is mixed with cells is 15-30% and thefinal content of hyaluronic acid is 2-4%.

If the chitosan fiber is added to the mixed polymer of the abovepluronic acid and hyaluronic acid in the present invention, it ispreferable to add and mix the same amount of the chitosan fiber to andwith the above mixed polymer.

Illustrated below are the method of manufacture of the composition forthe treatment of articular cartilage damage and the method of treatmentof articular cartilage damage using the above of the present invention.

The method of manufacture of the composition for the treatment ofarticular cartilage damage of the present invention is comprised of thesteps of collection of the umbilical cord blood; separation, culturing,and/or differentiation of mesenchymal stem cells from the umbilical cordblood; and mixing those mesenchymal stem cells and polymers. Each stepis further described in detail as follows:

In the step of collection of the umbilical cord blood, in case of thenormal vaginal delivery, the umbilical cord blood is collected from theumbilical vein that is extracted fully to the outside in a state thatthe placenta remains in the uterus after childbirth, or from theumbilical vein in a state that the placenta is extracted fully from theuterus also after childbirth in case of cesarean section.

When collecting the umbilical cord blood from the umbilical veinextracted fully to the outside of the uterus after childbirth in thepresent invention, it is collected from the umbilical vein connectingthe placenta and fetus after an infant is born according to aseptis,where both methods of collecting the umbilical cord blood before theplacenta is removed in the uterus after childbirth and of collecting theumbilical cord blood externally after the placenta is removed may beused. After the umbilical vein is secured, the umbilical cord blood iscollected in a collection bag containing an anticoagulant by usingcollection needles.

All conventional methods including that in Korean Patent Application No.10-2002-0008639, and those in Pittinger M F, Mackay A M, et al., Science1999; 284: 143-7, Lazarus H M, Haynesworth S E, et al., Bone MarrowTransplant 1995; 16: 557-64 may be used for the method of separation andculturing of mesenchymal stem cells and mesenchymal stem/progenitorcells from the umbilical cord blood collected as described in the above.Among them, an example is described as follows:

Mononuclear cells are separated through centrifugal separation of theumbilical cord blood and washed several times in order to remove foreignmaterials. If they are plated and cultured in a culture dish and/orflask and/or other container after they are washed, cells areproliferated with forming monolayer. Among them, mesenchymal stem cellsand/or stem/progenitor cells are those of which shape observed through ainverted microscope is homogeneous and that are proliferated in the formof colonies of long cells of the spindle shape. Thereafter, when thecells are grown to be confluent, sub-culturing is performed in order tohave the cells proliferated until the necessary number of cells isreached.

Mesenchymal stem cells and stem/progenitor cells originated from theumbilical cord blood of the present invention may be used directly forthe operation or after they go through the differentiation process.

As to the method of differentiation of mesenchymal stem cells originatedfrom the umbilical cord blood of the present invention, any propermethod in which desired cells may be obtained may be selected and usedamong conventionally used methods (Barry F, Boynton R E, et al., Expcell Res 2001; 268: 189-200, Jaiswal N, Haynesworth S E, et al., J CellBiochem 1997; 64: 295-312). Among them, one example is described asfollows:

While culturing cells originated from the umbilical cord blood in properchondrogenic differentiation media or osteogenic differentiation media,to what degree differentiation is progressed is confirmed through themeasurement of expression of enzymes, immune expression type analysis,histochemical stain, histoimmunologic stain, molecular biologicalexamination, or cellular medium analysis. Mesenchymal stem cells thusmanufactured and the cells differentiated from them may be used directlyfor the operation, or may be kept frozen, thawed when necessary, andproliferated again to be used.

The method of keeping the cells of the present method frozen isperformed according to the widely known method (Doyle et al., 1995). Themedium used for keeping frozen is composed of 10-20% FBS (fetal bovineserum), 10% DMSO (dimethylsulfoxide), and 5-10% glycerol. The cells aresuspended in such a way that about 1×10⁶ to 5×10⁶ cells exist in 1 ml ofthe above medium.

The above suspension of cells is distributed into glass or plasticamples for low-temperature freezing, sealed, and put into a controlledrate freezer with the conditions for temperature adjusted in advance. Itis preferable to use a freezing program offering the change oftemperature of −1° C./min when freezing the cells since it is possibleto reduce damage to the cells when they are thawed thereafter. Once thetemperature of amples reaches −180° C., they are transferred to a liquidnitrogen storage tank. The cells kept frozen may be stored for severalyears. Whether the viability of the cells is maintained should bechecked periodically at least every five years. When thawing the cellskept frozen, the amples are moved promptly to a water tub of whichtemperature is adjusted to 37° C. from the liquid nitrogen storage tank.The content thawed in the amples is moved immediately to a culturingcontainer having the medium containing 10% FBS and 5% ES in thesterilized state. The density of cells on the culturing medium isadjusted to have about 3×10⁵ to 6×10⁵ cells existed per ml of themedium. Whether the cells are proliferated is checked with a invertedmicroscope every day. When a proper density of cells is reached, thecells are transferred to a new medium for sub-culturing.

The cells cultured as described in the above are transplanted directlyto the region of articular cartilage damage in the state suspended on aproper medium or after they are mixed with polymers. In other words, amethod of injection of the cells suspended on the medium to a space madeof appropriate biomembranes such as periosteum, etc. of the region ofcartilage damage in a state not mixed with polymers, sealing them sothat the suspension of cells is not leaked out through the cracks ofthis periosteum, and suturing all of incised sections of operation maybe used. Or the above cells may be used by mixing them with appropriatepolymers along with the medium and making them in a state which issimilar to that of ointments or pastes. In all cases, it is preferableto adjust the concentration of cells contained finally in thecomposition of the present invention to have 1×10⁶ to 5×10⁷ cellsexisted in 1 ml. The amount of administration of the composition of thepresent invention may be increased or reduced according to the size ofthe portion of articular damage to be cured, and generally, it ispreferable to use about 2 ml in case of knee joints of adults having thesize of about 2 cm².

In the method of treatment of the articular cartilage damage using thecomposition of the present method, the articular cartilage portion to beoperated is observed preferably through the arthroscopic operation, thedamaged portion is prepared to facilitate the operation, the compositionof the present invention is applied to the damaged portion, and whetherit is positioned stably is confirmed preferably by using an arthroscope.The form of the composition of the present invention may be formulatedto fit into the damaged portion in advance before it is applied to thelesion or changed to fit after it is applied into the damaged portion.

As described above, the method of treatment of the articular cartilagedamage using the composition of the present invention may secure theconvenience of the procedures and reduce pain, sequelae and morbidity ofa patient since the damaged cartilage may be cured sufficiently throughthe arthroscopic operation, whereas conventional cell plantation methodsrequire for the operation of several times.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

The foregoing and other objects, aspects, and advantages will be betterunderstood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

FIGS. 1 a and 1 b are the diagrams in which the viscosity and strengthof a polymer manufactured by mixing pluronic acid and hyaluronic acid,and that manufactured by mixing the chitosan fiber, pluronic acid, andhyaluronic acid are confirmed;

FIGS. 2 a and 2 b are the diagrams showing the results of treatment ofthe portion of articular cartilage damage of rabbits by using thecomposition of the present invention; and

FIGS. 3 a and 3 b are the diagrams showing the amplified portion ofcartilage produced newly by the composition of the present invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

Hereinafter, the present invention is illustrated in more detail in apreferred embodiment of the invention. However, the scope of the presentinvention is not limited by that preferred embodiment, and it could bechangeable for human application.

Referring now to the drawings, illustrated in the following preferredembodiment are the methods of manufacture of the composition of thepresent invention and of treatment of the articular cartilage damageusing the above.

Firstly, cellular components are separated and cultured as follows inorder to manufacture the composition of the present invention:

Mononuclear cells are separated through centrifugal separation of theumbilical cord blood and washed several times in order to remove foreignmaterials. If they are plated and cultured in a culture dish and/orflask and/or other container after they are washed, cells areproliferated with forming monolayer. Among them, mesenchymal stem cellsand/or stem/progenitor cells are those of which shape observed through ainverted microscope is homogeneous and that are proliferated in the formof colonies of long cells of the spindle shape. Thereafter, when thecells are grown to be confluent, sub-culturing is performed in order tohave the cells proliferated until the necessary number of cells isreached.

Mesenchymal stem cells and/or stem/progenitor cells of the umbilicalcord blood obtained as described in the above are treated with trypsin,washed, suspended on a DMEM medium, and prepared for so that they may bemixed with the polymer in subsequent steps.

Secondly, for the manufacture of the composition of the presentinvention, polymers are prepared as follows and chemical, biologicaland/or mechanical properties of polymers are confirmed by performingexperiments confirming their chemical, biological and/or mechanicalstrength in advance.

In order to confirm the mechanical strength according to theconcentration or addition or reduction of the components comprisingpolymers, a polymer containing 30% of pluronic acid finally, a polymercontaining 4% of hyaluronic acid finally, a polymer containinghyaluronic acid of each different concentration while containing 15% ofpluronic acid finally, and a polymer containing the mixture of the abovepluronic acid and hyaluronic acid and further containing the same amountof the chitosan fiber are prepared, in which the final concentrations ofhyaluronic acid are 0.5%, 1.0%, 1.5%, 2.0% and 4.0%, respectively.

Each polymer is prepared by mixing the above components and whether eachhas the proper/appropriate property is evaluated.

As a result, as shown in FIG. 1 a, a fairly good mechanical strength isshown in case of the polymer containing hyaluronic acid of eachdifferent concentration while containing 15% of pluronic acid finally.But it is seen that the mechanical strength is lowered as theconcentration of hyaluronic acid is lowered. Particularly, it isobserved that the polymer flows down along the wall of the tube if thetube is held upside down when less than 0.5% of hyaluronic acid iscontained finally as the mechanical strength is lowered significantly.

On the other hand, the polymer containing the mixture of the abovepluronic acid and hyaluronic acid and additionally the same amount ofthe chitosan fiber shows the flexibility of the degree which is good tochange the shape when it is applied to the damaged region whilemaintaining the mechanical shape irrespective to the amount ofhyaluronic acid. The polymer containing 30% of pluronic acid finallyalso shows proper mechanical strength and flexibility.

Accordingly, it is possible to select a polymer having the appropriatestrength and flexibility to the degree which is proper for applying tothe treatment of the region of cartilage damage and its mixedcomposition.

Thirdly, in order to manufacture the composition of the presentinvention, among the polymers manufactured in the above, for example, apolymer containing 30% of pluronic acid finally is selected, of which0.3 g is mixed with about 0.9 ml of the DMEM medium sufficiently.Thereafter, the suspension of cells in the first step in which 1×10⁷cells are contained is concentrated in terms of centrifugal separation,the supernatant is discarded, and the polymer made in the second step isput into the cell portion only. A proper amount of the medium is thenadded to the above to make the final volume of 1 ml. That is, thecomposition for the treatment of articular cartilage damage of thepresent invention is manufactured to include 30% (0.3 g) of the polymerand 1×10⁷ cells in 1 ml of the medium. If the amount becomes greater,they are mixed at the same ratio.

Fourthly, the articular cartilage damage is treated by using thecomposition for the treatment of articular cartilage damage of thepresent invention.

In order to design models for the articular cartilage damage, a healthyrabbit is selected and proper amounts of ketamine (35 mg/kg) andxylazine (5 mg/kg) according to its body weight are injectedintramuscularly. After it is confirmed that the anesthesia of the rabbitis completed fully, the knee joints of both legs are shaved and fixedwith an adhesive plaster while maintaining the supine posture. Both kneejoints are disinfected with betadine, their locations are confirmed bypalpatiom with fingers, after which the inside of the joint is observedby reaching the inside of knee joints through the paramedian approachalong the incision line passing through the upper and lower portions ofknee joints and inner side of the knee cap and bending the knee jointswhile pushing the knee cap toward the outer side.

After it is confirmed that there are no particular pathologic findings,a centralizing dimple is made at a position 4 mm above the front upperend of the central interchondylar notch of the distal femur with asharp-pointed gimlet and a hole having the diameter of 3 mm and depth of3 mm is made centering around the scar with a drill. A punch having thediameter of 3 mm may be used for the same region, and the full thicknesscartilage defect is created. The damaged region is observed 3-4 monthsafter the cartilage damage is induced as in the above. And it isconfirmed that the damaged region of cartilage is not cured by itself.

In the meantime, 0.25 ml of the composition of the present inventionmanufactured in the third step is injected and pushed into the damagedregion of cartilage of a rabbit by using a syringe.

Thereafter, the knee cap is returned to its original position, softtissues around the knee cap are repaired with absorbable sutures, andthe skin is closed with non-absorbable sutures. The leg on the oppositeside is regarded to be a control group, into which only biodegradablepolymers are applied.

After it is confirmed that the rabbit regains consciousness fromanesthesia, it is allowed to move freely, and antibiotics areadministered to it in order to prevent infection after the operationuntil the next day. After 11 weeks, sections of the articular cartilageregion of each rabbit which were subject to damage and treated areobtained and inspected for newly formed cartilage. The results ofcomparison are shown in FIGS. 2 a and 2 b.

As shown in FIGS. 2 a and 2 b, the total thickness of repaired layersproduced newly after the composition of the present invention is appliedis shown to be more than twice greater (FIG. 2 b) than that when onlythe polymer is applied (FIG. 2 b).

FIGS. 2 a and 2 b are amplified and shown in FIGS. 3 a and 3 b in orderto compare the property of newly formed cartilage repair tissue. Asshown in FIGS. 3 a and 3 b, cartilage tissue of the almost sameappearance as that of original normal outside cartilage tissue (rightside) are produced newly (left side) if the composition of the presentinvention is applied (FIG. 3 b), although the cellular density isgreater than that of original normal cartilage tissues. On the otherhand, if only the polymer is applied (FIG. 3 a), the shape of cells isrough compared to that of normal cells and the density is shown to below.

Accordingly, it is seen that the composition of the present invention isable to produce cartilage tissue in the damaged region of articularcartilage with a superior efficiency and to treat the articularcartilage damage effectively.

In conclusion, the composition for the treatment of articular cartilagedamage of the present invention shows excellent effects in histologicaspects for the treatment of the cartilage damage. Compared to theconventional methods of treatment of the articular cartilage damage, themethod of its treatment using the composition of the present inventionis able to reduce the time, efforts, and expenses for the treatment ofarticular cartilage damage since the subject method has an effect ofsufficiently repairing the articular cartilage damage by employingsimple procedures such as the arthroscopic operation or much simpleroperation.

While the invention has been described in terms of a single preferredembodiment, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

1-14. (canceled)
 15. A composition comprising osteocytes and/orosteoblasts differentiated from mesenchymal stem cells, wherein themesenchymal stem cells are isolated from umbilical cord blood.
 16. Thecomposition according to claim 15, wherein the mesenchymal stem cellsare further proliferated.
 17. The composition according to claim 15,which treats bony damage, loss, or defects when administered to a siteof bony damage, loss, or defects in a subject.
 18. The compositionaccording to claim 15, which is in an injectable formulation.
 19. Thecomposition according to claim 15, wherein said composition contains1×10⁶ to 5×10⁷ cells per mL of the composition.